Salmonellae and pollution indicator bacteria in municipal and food processing effluents and the Cornwallis River

A study was conducted to determine the incidence of fecal coliforms, fecal streptococci, Aeromonas hydrophilia, and Salmonella spp. in the waste discharges of seven sewage treatment plants, four fruit and vegetable canneries, a meat packing plant, a poultry processing plant, and a potato processing plant located along the Cornwallis River in Nova Scotia, Canada. Surface water samples were also collected from 13 locations in the river to assess the impact of these waste discharges on the receiving water quality. The results showed that the final effluents from most of the sewage treatment and processing plants were of very poor bacteriological quality, with the number of indicator bacteria comparable with those found in raw sewage. Salmonellae were isolated from the effluents of the meat and poultry plants and five of the seven sewage treatment plants surveyed. No salmonellae were detected in the effluents of the fruit and vegetable canneries. The impact of the discharge of untreated municipal and food processing wastes on the Cornwallis River water quality was evidenced by the recovery from river water of five Salmonella serotypes, and the high fecal coliform counts which exceeded recommended limit for bathing and shellfish harvesting.     

Potential Regrowth and Recolonization of Salmonellae and Indicators in Biosolids and Biosolid-Amended Soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Bacteriology of Dehydrated Space Foods

The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count ( 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive.     

Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers

Very little is known about the microbial composition of animal bedding wastes, including poultry litter, and what is known has been deduced from standard culture methods, by which some fastidious organisms that exist in the environment may not be detected. We evaluated the bacterial composition of poultry litter by using a combination of culture and molecular detection. Total aerobic bacteria in poultry litter were detected by culture at 10(9) CFU/g of material. Enteric bacteria such as Enterococcus spp. and coliforms composed 0.1 and 0.01%, respectively, of the total aerobic cultivatable bacteria in poultry litter; no Salmonella strains were detected by culture. In order to characterize the most abundant bacterial groups, we sequenced 16S ribosomal DNA (rDNA) genes amplified by PCR with microbial community DNA isolated from poultry litter as the template. From the 16S rDNA library, 31 genera were identified. Twelve families or groups were identified with lactobacilli and Salinococcus spp. forming the most abundant groups. In fact, 82% of the total sequences were identified as gram-positive bacteria with 62% of total belonging to low G+C gram-positive groups. In addition to detection of 16S rDNA sequences associated with the expected fecal bacteria present in manure, we detected many bacterial sequences for organisms, such as Globicatella sulfidofaciens, Corynebacterium ammoniagenes, Corynebacterium urealyticum, Clostridium aminovalericum, Arthrobacter sp., and Denitrobacter permanens, that may be involved in the degradation of wood and cycling of nitrogen and sulfur. Several sequences were identified in the library for bacteria associated with disease in humans and poultry such as clostridia, staphylococci, and Bordetella spp. However, specific PCR targeting other human and veterinary pathogens did not detect the presence of Salmonella, pathogenic Escherichia coli, Campylobacter spp., Yersinia spp., Listeria spp., or toxigenic staphylococci. PCR and DNA hybridization revealed the presence of class 1 integrons with gene cassettes that specify resistance to aminoglycosides and chloramphenicol. Only from understanding the microbial community of animal wastes such as poultry litter can we manage animal disease and limit the impact of animal waste on the environment and human and animal health.     

Microbiological Quality of Protein Feed Supplements Produced by Rendering Plants

Samples of protein feed supplements produced by rendering plants were examined for salmonellae, total aerobic bacterial counts, coliform counts, and enterococci. Isolations of salmonellae were more frequent from products with high counts; however, 6% of the samples with total counts of less than 1,000 per gram and 14% of the samples with coliform counts of less than 1 per gram contained salmonellae. Serotypes of Escherichia coli which have been associated with disease in domestic animals and poultry were also isolated from products. Although the distribution of serotypes of salmonellae isolated from environmental swabs and flies was similar to that isolated from products, the isolation of several serotypes from flies which had not been isolated in plants suggested that flies may be a potential source of contamination.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Automated electrical impedance technique for rapid enumeration of fecal coliforms in effluents from sewage treatment plants.

Fecal coliforms growing in a selective lactose-based broth medium at 44.5 degrees C generate a change in the electrical impedance of the culture relative to a sterile control when populations reach 10(6) to 10(7) per ml. The ratio of these changes was measured automatically, and the data were processed by computer. A linear relation was found between the log10 of the number of fecal coliforms in an inoculum and the time required for an electrical impedance ratio signal to be detected. Pure culture inocula consisting of 100 fecal coliforms in log phase or stationary phase were detected in 6.5 and 7.7 h, respectively. Standard curves of log10 fecal coliforms in wastewater inocula versus detection time, based on samples collected at a sewage treatment plant over a 4-month period, were found to vary from one another with time. Nevertheless, detection times were rapid and ranged from 5.8 to 7.9 h for 200 fecal coliforms to 8.7 to 11.4 h for 1 fecal coliform. Variations in detection times for a given number of fecal coliforms were also found among sewage treatment plants. A strategy is proposed which takes these variations into account and allows for rapid, automated enumeration of fecal coliforms in wastewater by the electrical impedance ratio technique.     

Bacterial pollution indicators in the intestinal tract of freshwater fish

A study was made of the occurrence, distribution, and persistence of coliforms, fecal coliforms, and fecal streptococci in the intestinal tract of freshwater fish. A total of 132 fish representing 14 different species were used in various phases of these experiments. Examination of the intestinal contents of 78 fish from moderately polluted sections of the Little Miami River indicated that fecal coliform densities were lowest in bluegills (less than 20 per gram) and highest in catfish (1,090,000 per gram). Levels of fecal streptococci for these two species were 220 and 240,000 per gram, respectively. The occurrence of fecal coliforms in fish caught in this stream reflected the warm-blooded-animal-pollution level of the water. All fish used in this phase of the study were caught during July, August, and September when the water temperatures were between 13 and 18 C. The fate of fecal coliforms and Streptococcus faecalis in the fish intestine indicated that these organisms can probably survive and multiply when fish and water temperatures are 20 C or higher, but only when the organisms are retained in the gut for periods beyond 24 hr. Based on the biochemical reactions for 3,877 coliform strains isolated from 132 freshwater fish of 14 different species, 91.4% of all strains were composed of five IMViC types. In a similar study of the biochemical reactions of 850 streptococci isolated from the intestinal tract of 55 freshwater fish, the predominant strains included S. faecalis and various closely associated biotypes. No consistently recurring pattern for either coliforms or streptococci could be developed to identify species of fish investigated. The composition of the intestinal flora is, however, related in varying degree to the level of contamination of water and food in the environment.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Salmonellae in Fish Meal Plants: Relative Amounts of Contamination at Various Stages of Processing and a Method of Control

Previous studies have shown that Menhaden fish meal, a common ingredient of animal feeds, is frequently contaminated with salmonellae. Animals that eat contaminated feed may become infected. If they, in turn, are eaten by humans, they may be a means by which salmonellae are introduced into the human population. Epidemiological studies of the fish-meal industry were carried out to determine the sources of salmonellae in fish meal and the factors affecting the persistence and survival of salmonellae during the processing of fish meal. Examination of 190 fish immediately after they came from the Gulf of Mexico revealed no salmonellae, but salmonellae were frequently isolated from samples of fish taken from the boats when they arrived at the plants. Salmonellae were also frequently isolated from dockside water at each of the plants. Approximately 50% of the samples taken in the raw fish processing areas were contaminated with salmonellae. The percentage of samples yielding salmonellae decreased progressively through the various sequences of processing, but more than 15% of the samples taken from the finished products were also positive. Salmonellae were isolated from the raw area of the plant most frequently while the plant was operating and less frequently when the plant was idle, whereas in the processing area of the plant the reverse was true. Salmonellae appeared to survive and multiply in the processing area of the plant while the plant was idle, which resulted in contamination of the first portion of each day's production. Salmonellae in the processed fish meal were reduced to nondetectable levels by reprocessing the first 45 min of each day's production.     

Value of waste water in agriculture. Evaluation of soil bacterial contamination, water table and plant culture]

The present study aim was to valorize the treated waste water as source of fertilizers for vegetables seed production and to assess the eventual bacteriological contamination risks of soil, plant and phreatic ground water table. The bacteriological analysis of drained water did not reveal any fecal coliforms vertical migration in depth and a low fecal contamination (thermotolerant coliforms) is limited to the levels of superficial horizons. The seed produced by using waste water showed a slightly fecal contamination which disappeared following treatment with a (5% chloride solution. The treated waste water improve the onion seeds production per hectare in spite of the increases of the phytopathogenic hazards.     

Persistence and distribution of pollution indicator bacteria on land used for disposal of piggery effluent.

Numbers of pollution indicator bacteria (fecal coliforms and fecal streptococci) were assessed on land to which effluent from intensively housed pigs had been applied. Topsoil (to a 30-mm depth) was found to provide a more favorable environment for fecal coliform persistence than was pasture or subsoil. Times required for a 90% reduction in number (T90) in topsoil (calculated by linear regression of log counts obtained in a 6-week period after effluent application) ranged from 7 to 20 days (mean T90, 11 days). T90 values for fecal coliforms fell within this range irrespective of the season of application and for a number of soil types and climatic conditions. The range in die-off times was encountered irrespective of the fecal coliform count in the applied effluent or the application regimen (125 to 1,000 kg of elemental nitrogen in the form of effluent per ha; return periods, 3 to 12 months). Autumn and winter conditions were conducive to the persistence of a survivor tail of these bacteria at 10(1) to 10(3) cells per g of topsoil. Fecal streptococci survived similarly on soil and pasture (T90, ca. 14 days) and appeared slightly more suited to survival in the environment than did fecal coliforms. Contamination of subsoils after effluent applications occurred at a rate well in excess of the infiltration capacity of the soil, presumably by percolation of the effluent through soil cracks. Contamination levels of subsoils in the experimental area generally remained low.     

Coliform concentrations in Lake Erie — 1966 to 1970

Total coliform concentrations obtained from cruises of Lake Erie, conducted from the Canada Centre for Inland Waters between 1966 and 1970, have been analysed to determine the spatial and temporal distribution of total coliforms in the lake. Although year to year comparisons were made on a qualitative level due to the limitations in the data, some consistent seasonal features of the spatial distribution are evident. The location of zones of higher concentration have been attributed to proximity to large urban areas and lake currents. The contribution of fecal coliforms to the total coliform concentration has been examined with the conclusion that, at the low total coliform concentrations ( 30 per 100 ml) observed in much of the lake, there is no relationship between the two concentrations but, over a broader range, fecal coliforms increase with total coliform concentrations and, at very high total coliform concentrations (1000 per 100 ml), fecal coliforms are always present. The analysis demonstrates 1) the value of examining observed and fitted distributions to compare subsets of a large collection of data and 2) a clustering procedure which is generally applicable to data consisting of counts.     

Increase in microbial contamination of defeathering machinery in a poultry processing plant after changes in the method of processing

The numbers of coliforms, Enterobacteriaceae and Staphylococcus aureus and the extent of their colonization of the fingers of the defeathering machinery within a poultry processing plant were increased after changes in machinery design and a reduction in the level of chlorination. Such increases and the pattern of contamination suggested that the changes had allowed an endemic flora to develop on the fingers.     

Incidence of Salmonellae in Animal Feed and the Effect of Pelleting on Content of Enterobacteriaceae

During a survey of meat and bone meals, fish meals and poultry feeds salmonellae were found only in samples of meat and bone meal, for which 19% were Salmonella -positive. MPN estimations on 2 meat and bone meal samples gave figures of 6 and 39 salmonellae/ 100 g, respectively. Sampling of poultry feed before and after pelleting showed that this process gave up to 1000-fold reduction in numbers of Enterobacteriaceae depending on the type of processing involved.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish.

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.     

Relationship among fecal coliforms, Escherichia coli, and Salmonella spp. in shellfish

The relationship of fecal coliforms, Escherichia coli, and Salmonella spp. was examined in freshly harvested and stored shellfish. In 16 of 40 freshly collected oyster samples, fecal coliform levels were above the recommended wholesale level suggested by the National Shellfish Sanitation Program (less than or equal to 230/100 g), and Salmonella spp. were present in three of these samples. Salmonella spp. were not, however, present in any sample containing less than 230 fecal coliforms per 100 g. Analysis of the data suggests that low fecal coliform levels in both fresh and stored oysters are good indicators of the absence of Salmonella spp., but that high levels of fecal coliforms are somewhat limited in predicting the presence of Salmonella spp. E. coli levels correlated very strongly with fecal coliform levels in both fresh and stored oysters and clams, suggesting that there is no advantage in replacing fecal coliforms with E. coli as an indicator of shellfish quality.